Fig 1.
Modular strategy used for the construction of pFA plasmids with recyclable markers.
(A) Construction of pFA deletion plasmids. The five recyclable markers flanked by loxP repeats were amplified with oligonucleotides LXL1 and LXL2, which generated overlapping ends with the pFA-CaHIS1 backbone digested with BamHI and PmeI. The amplified fragments were assembled with the vector in 5 independent reactions using NEBuilder HiFi DNA Assembly kit. (B) Construction of epitope-tagging plasmids. The different tagging modules (GFPγ, 3xGFPγ, mCherry, 3xHA, 5xmyc or TAP-TAG) were amplified with oligonucleotides that generated overlapping ends with the pFA-CaHIS1 backbone digested with BamHI (red) and with the 5´end of the recyclable marker module (light blue). The five recyclable markers were independently amplified with oligonucleotides that produced DNA fragments containing overlapping ends with the tagging modules (light blue) and the pFA-CaHIS1 backbone digested with PmeI (yellow). The different modules were assembled with the vector in independent reactions using NEBuilder HiFi DNA Assembly kit.
Table 1.
Plasmids generated and fragment length of the cassettes.
Table 2.
Primers used.
Fig 2.
Construction of deletion mutants without selection markers.
(A) The sep7::LUL/sep7::NAT1-Clox strain (OL2908) was grown overnight in YPD medium with (+M/C) or without (-M/C) methionine and cysteine to repress or induce the expression of the Cre recombinase respectively, and then plated on YPD medium. After 24 h of incubation at 28ºC, the plate was printed onto YPD+ nourseothricin or SC -Uri plates and incubated another 24 h. (B) PCR analysis of the different strains with specific SEP7 oligonucleotides. Genomic DNA was isolated from the parental strain BWP17 (1) and the sep7::loxP/sep7::loxP (2, OL2909) mutant and analyzed with two different oligonucleotide pairs, one annealing at the 5´ and 3´ flanking regions (A and B) and another internal to the coding region (C and D) to confirm correct integration and excision of the deletion cassettes. The position of the primers in the SEP7 locus and the sep7::loxP allele is depicted to the left.
Fig 3.
Generation of auxotroph strains containing three genes tagged with GFPγ.
(A) Growth in selective media. Single colonies of strains BWP-17, NUP49-GFPγ::LAL/NUP49 (OL2845), NUP49-GFPγ::LAL/NUP49 TUB2-GFPγ::LUL/TUB2 (OL2847), NUP49-GFPγ::LAL/NUP49 TUB2-GFPγ::LUL/TUB2 CDC12-GFPγ::NAT1-Clox/CDC12 (OL2867) and the resolved strain NUP49-GFPγ::loxP/NUP49 TUB2-GFPγ::loxP/TUB2 CDC12-GFPγ::loxP/CDC12 (OL2866) were grown on YPD plates and replica-printed to -Arg, -Uri and YPD + nourseothricin media. Plates were incubated 2 days at 28ºC. (B) Images of cells carrying Nup49-GFPγ, Tub2-GFPγ and Cdc12-GFPγ during yeast growth. Exponential growing cultures of the NUP49-GFPγ::loxP/NUP49 TUB2-GFPγ::loxP/TUB2 CDC12-GFPγ::loxP/CDC12 strain (OL2866) were imaged. The images are the maximum projection of 5 planes and show the differential interference contrast image (DIC), the GFP channel and the merged image (DIC in red, GFP in green). Scale bar, 2 μm. (C) Comparison of GFPγ and 3xGFPγ intensity. Images of CDC12-GFPγ::LAL/CDC12 or CDC12-3xGFPγ::LAL/CDC12 cells during yeast growth. The images are the maximum projection of 3 planes and show the differential interference contrast image (DIC, in red) and the GFP signal (green). Scale bar, 2 μm. The graph represents the average intensity of the signal ± s.e.m. in both strains. At least 50 single rings were measured in each strain.
Fig 4.
Double fluorescent tagging with GFPγ and mCherry.
(A) Growth in selective media. Single colonies of strains BWP-17, CDC12-mCherry::LAL, CDC12-mCherry::LAL TUB2-GFPγ::URA3-Clox and two independent clones of the resolved strain CDC12-mCherry::loxP TUB2-GFPγ::loxP (1 and 2) were grown on YPD plates and replica-printed to SC -Arg, SC -Uri or SC -Arg/-Uri media. (B) Localization of Cdc12-mCherry and Tub2-GFPγ during yeast growth. Exponential growing cultures of the CDC12-mCherry::loxP TUB2-GFPγ::loxP strain were imaged. The images are the maximum projection of 5 planes and show the differential interference contrast image (DIC), the Tub2-GFPγ and the Cdc12-mCherry channels, and the merged image (DIC in red, Tub2-GFPγ in green and Cdc12-mCherry in blue). Scale bar, 2 μm.
Fig 5.
(A) Growth in selective media. Single colonies of strains SEP7-HA::LAL/SEP7 (2), SEP7-HA::LAL/SEP7 CDC10-HA::LHL/CDC10 (3), SEP7-HA::LAL/SEP7 CDC10-HA::LHL/CDC10 CDC12-HA::NAT1-Clox/CDC12 (4) and SEP7-HA::loxP/SEP7 CDC10-HA::loxP/CDC10 CDC12-HA::lox/CDC12 (5) were grown on YPD plates and replica-printed to SC -Arg, SC -His or YPD+ nourseothricin media. (B) Western blot analysis of the same strains using anti-HA antibody. The BWP17 parental strain (1) was included as an untagged control. Protein extracts were separated on 4–12% Bis-Tris gels, blotted to PDVF membranes and incubated with anti-HA antibody.